Various methods are presently used for the qualitative and/or quantitative determination of specific binding agents and/or their corresponding bindable substances. Although these methods differ widely from each other in sensitivity, ease of operation and chemical and physical principles involved, important similarities are generally recognized. Typical examples of the relationship between a specific binding agent and its corresponding bindable substance(s) are of the type antigen-antibody, antibody-antigen, protein-protein, protein-ligand, receptor-ligand or nucleic acid-complementary nucleic acid. Antigen-antibody or immunological interactions are by far the most important in this connection, and particularly for diagnostic purposes, detection methods based on such interactions are the most widely used today.
Various techniques can be employed to detect and optionally quantify the aggregates, by aggregates we mean complexes formed between the specific binding agents and bindable substances involved. In certain instances, the complexation reaction will lead to a directly visible signal as a result of agglutination and/or precipitation of the aggregate itself. This will however not always be the case and, in general, the concentration of binding agent and bindable substance, needed to produce such result, will be far above the practical and useful limits. In order to circumvent this lack of sensitivity or to detect otherwise un-detectable aggregates, various methods have been developed such as, for example, complement fixation, passive haemagglutination, radio-immuno assay (RIA), immuno-fluorescence and enzyme-linked immuno sorbent assay (ELISA). In the last three methods, the detection of the aggregate is improved by labelling the aggregate with an easily detectable marker, which is either bound directly to the specific binding agent, to a secondary binding agent for which the primary binding agent acts as a bindable substance, or to the bindable substance. In the three methods listed, the marker is respectively a radioactive atom or group, a fluorescent substance or an enzyme. Such methods are described i.a. in Weir's Handbook of Experimental Immunology (1967), Blackwell Scientific Publications, Oxford and Edinburgh and U.S. Pat. No. 3,654,090 (ELISA).
During the last years, methods have been introduced wherein aggregates formed between specific binding agents and bindable substances are detected by labelling the said aggregates directly or indirectly with small sized metal particles, particularly gold particles. Depending on the circumstances, these particles can be detected, e.g. by direct visual examination, by microscopic or spectrophotometric techniques. A description of the "immunogold staining (IGS) technique", "the sol particle immuno assay (SPIA) technique" of specific applications and improvements thereof can be found e.g. in U.S. Pat. Nos. 4,313,734, 4,446,238 and 4,420,558, in U.S. Ser. No. 622,923, which corresponds to the European Patent Publication No. 165,634, in U.S. Ser. No. 660,832, which corresponds to the Eur. Pat. Publ. No. 158,746 and in IBRO handbook series, Wiley, New York, 1983, pages 347 to 372.
Metal particles have further been employed for the staining of acceptor substances, such as proteins and nucleic acid, which are directly immobilized on a solid support. Such a method is for example described in U.S. Ser. No. 744,091, which corresponds to the European Patent Publication No. 165,633.
Starting from a relatively unknown method for labelling cell surface antigens, metal particles have today become widely used in a variety of detection and/or quantitive determination problems. The possibility of direct visual examination of metal particles and the advantage that the signal generated is permanent and not prone to rapid degradation makes it an interesting marker for simple and rapid assays. Moreover metal markers, preferably gold markers, seem preferable over radioisotope markers due to the very low health hazard related to working with the former.
In the European Patent Publication No. 158,746 page 10 lines 18 to 32 there is described a method to improve the signal of a colloidal gold marker significantly by subjecting the colloidal gold particles bound to the surface of a blotting medium to a so-called physical developing procedure.
The art-known physical developers generally consist of a solution containing a soluble metal salt, such as silver nitrate, a reducing agent, such as hydroquinone and an appropriate buffer system to establish a specific pH, preferable less than pH 4.
Initially the reduction of silver ions to metallic silver is catalyzed at the surface of gold particles resulting in a specific deposition of metallic silver at the gold particle site. In turn, the thus formed metallic silver particles catalyze the reduction, creating an auto-catalytic process. The effect of a physical development is that the reddish optical gold signal turns into a deep-brown to black silver signal, with a much higher intensity. The use of these art-known physical developers results in an improved signal, although there are a number of drawbacks associated with it.
One of the major problems is the solubility of the metal salts. Indeed, it is well known that metal ions, such as silver ions, form insoluble salts with many counter ions. Apart from depleting the available silver ion supply, these insoluble salts also form nuclei at which the reduction process is catalyzed as well, which results in a seriously augmented noise level. Moreover, silver ions may form light sensitive silver salts, such as silver bromide and silver chloride, which are readily reduced to metallic silver under the influence of light, starting an auto-catalytic process. It is therefore absolutely necessary to work with extremely clean contacting surfaces, e.g. vessels, analytical grade chemicals and ultra-pure water. Usually it is also necessary to introduce multiple washing steps between the incubation with the metal-marked specific binding agent and the physical development of the marker in order to remove unwanted ions present in the incubation medium. All this tends to make traditional physically developed metal-based assays more complex, expensive and error prone.
The major disadvantage of the traditional methods lies within the nature of physical developing itself. In the case of a silver-based physical developer, for example, the reducing agent reduces all silver ions at a certain rate. To obtain optimal sensitivity the amplification process has to be aborted by removing the physical developer from the metal marker-containing phase before the non-marker-induced reduction, the so-called `self-nucleation`, becomes apparent. It is obvious that physical developers become more flexible and powerful if the ratio between metal-specific reduction speed and speed of self-nucleation can be augmented. With the traditionally used physical developers, this ratio can hardly be augmented. The only parameter which can be modulated is the overall speed of the process; self-nucleation can be postponed only at the expense of a slower metal marker amplification. One of the most obvious ways to do this is to change the concentration, nature or environment of the reducing agent. A frequently used approach is the use of hydroquinone at a pH lower than 4. The reducing action of hydroquinone is strongly inhibited in an acid environment; the user of the physical developer therefore has enough time to stop the metal marker amplification before self-nucleation causes too much noise. However, acid additions are in many cases not compatible with the nature of the binding between the marked specific binding agent and its corresponding bindable substance. Most monoclonal antibodies have only a low or average affinity to their antigens at said pH. Moreover, no real gain in sensitivity can be accomplished because the marker amplification is slowed down to the same degree as the self-nucleation is slowed down.
Thus there is a strong need for improving the sensitivity and practicality of metal based detection and/or quantitative determination techniques.